Project Details
Projekt
Differential protein- and RNA-interactions of Roquin specify alternative modes of post-transcriptional gene regulation
Subject Area
General Genetics and Functional Genome Biology
Biochemistry
Structural Biology
Cell Biology
Biochemistry
Structural Biology
Cell Biology
Term
from 2016 to 2023
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 313381103
Appropriate immune responses require CD4+ T cells to make profound cell fate decisions, which are placed under post-transcriptional control. A key player in these decisions is the RNA-binding protein (RBP) Roquin. While the prototypic interaction of Roquin with its consensus decay element and subsequent deadenylation and decapping of the target mRNA is well understood, many observations imply a much higher complexity of Roquin-mediated gene regulation. It not only involves interactions with additional cofactors and different post-transcriptional effectors causing different modes of post-transcriptional regulation but also interactions with different recognition elements in mRNAs and composite binding sites. In the first funding period, we studied the post-transcriptional regulation of Icos mRNA by the RNA-binding protein Roquin-1. We could show that Roquin-1 directly interacts with its cofactor Nufip2 and thereby increases Icos mRNA repression. Additional co-regulators identified by us and colleagues include the endonuclease Regnase-1 and the deadenylation factor Cnot1. In the second funding period we will therefore characterize and quantify the interaction of Roquin with Nufip2, Regnase-1, and Cnot1, map their interaction surfaces in case of direct interactions, and perform structural analyses of their co-complexes by X-ray crystallography, micro electron diffraction, and NMR. We will use this information to generate mutations that specifically abolish a particular interaction, verify their loss of binding, and characterize in T cells their global effects on gene regulation. In the first funding period, we performed a saturating mutagenesis screen with over 10,000 distinct mutations in the 2.6 kb long 3‘-UTR of Icos mRNA and used next-generation sequencing to quantify the effects of mutations on Icos mRNA regulation. In the second funding period we will perform an in-depth analysis of the results of this mutagenesis screen. Cis-acting elements identified in the screen will be reassessed in T cells using the CRISPR/Cas9 technology for reproducibility of their regulatory function and to understand their specific modes of regulation. Furthermore, we aim to identify proteins binding close to these cis-elements by using a RNA-tethered proximity ligation approach. RNA-binding proteins and co-factors identified this way, will be assessed for their binding to the corresponding cis-acting element, as well as to Roquin, Regnase-1 and Nufip2. The importance and specific functions of the most relevant factors will be assessed in T cells by knock-out, followed by target gene analysis. In summary, by elucidating how Roquin-1 cooperates with different trans-acting co-factors to select specific modes of translational inhibition we aim at identifying principles of combinatorial post-transcriptional gene regulation of Roquin-dependent target mRNAs.
DFG Programme
Priority Programmes
